Abstract Non-viral gene delivery is used in most biomedical laboratories for basic research and for many commercial and medical applications. These include basic investigations into gene function, modification of cells for the production of recombinant proteins, and generation of genetically modified human cells for cancer therapy (e.g. chimeric antigen receptor transgenic T cells). However, the delivery of DNA by transfection for gene transfer is limited by its extremely high toxicity to many cell types, such as hematopoietic stem cells and other blood cell types (e.g. human T cells). Thus, gene delivery by DNA transfection is very inefficient except in a subset of cell lines selected for transfectability (e.g. HEK293T cells). In contrast, delivery of in vitro transcribed mRNA results in robust gene expression in a very high percentage of cells without toxicity, greatly out-performing DNA delivery. Unfortunately, the transient nature of an mRNA limits the utility of this gene delivery method to special, rare circumstances where a short duration of expression is acceptable. But in most gene therapy settings, and in many experiments, permanent gene expression is desired. In this proposal from B-MoGen Biotechnologies, Inc., we will establish the feasibiliy our entirely new gene delivery system in which an RNA molecule is delivered to cells, that is then converted to a DNA copy in the cell, where it is efficiently integrated into the genome and expressed. This novel system should combine the efficiency and non-toxicity of RNA gene delivery with the permanence of DNA gene delivery. The applications of this technology for research are many, including numerous settings where DNA transfection is sub-optimal. Moreover, this technology could revolutionize therapeutic gene delivery to human cells including correction of genetic diseases in blood stem cells, delivery of chimeric antigen receptor transgenes to T cells, and delivery of substrate DNA molecules for homology dependent repair after targeted nuclease-mediated double-stranded DNA cutting. The technology is based on a retrotransposon from mice, called the Intracisternal Type A Particle (IAP), which does not exist in human cells. As such, it is an ideal RNA-based permanent gene delivery vehicle for human cells and could be applied to tissues in situ.